The goal of this proposed research is to evaluate and validate the biological basis of emerging non-invasive imaging biomarkers for use in evaluation of treatment response in colorectal cancer (CRC). Current imaging criteria for evaluating therapeutic response are based upon anatomical information according to Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. These criteria, which are solely based on a reduction in tumor size, do not take advantage of cellular and molecular information now available through contemporary imaging methodology. Importantly, since relevant cellular and molecular changes may precede changes in size and occur within hours of treatment, RECIST criteria and conventional imaging methods are frequently inadequate for assessing early tumor response. These limitations, coupled with the increasing clinical relevance of employing complex, molecularly targeted therapeutic regimens to treat cancer, highlight a critical need to accelerate the translation of novel imaging approaches that are capable of reporting cellular and molecular responses of tumor cells to therapy. Presently, a major impediment to the clinical translation of novel imaging methodology is a lack of appropriate validation studies conducted within relevant biological contexts. The investigations proposed herein build upon extensive preclinical and clinical data accessible through our participation in both the Gastrointestinal Special Program of Research Excellence (GI SPORE) and the Mouse Models of Human Cancers Consortium (MMHCC) and aim to elucidate and validate the underlying biological factors and molecular events that affect specific cancer imaging biomarkers. In these studies, we propose comprehensive validation of three translational imaging metrics that report aspects of cellular proliferation and apoptosis, namely [18F]-FLT PET imaging, apparent diffusion coefficient mapping via MRI (ADC-MRI), and [99mTc]-Annexin-V SPECT imaging. Cellular proliferation and apoptosis are critical biological processes known to be dysregulated in cancer cells; thus non-invasive, longitudinal imaging assessments of these processes could be of particular value in predicting and quantifying response to therapy. Validation of the proposed imaging biomarkers will be performed within the context of molecularly targeted therapy for treatment of advanced CRC, a field where we have considerable Institutional experience within preclinical and clinical settings. To accomplish these goals, we have identified two specific aims. Aim 1- To explore quantitative relationships between non-invasive imaging biomarkers and discrete genomic and proteomic molecular events that regulate cell cycle and apoptosis in preclinical mouse models of human colorectal cancer. Aim 2- To explore the utility of registering three-dimensional, multi-parametric imaging data sets with small-molecule and proteomic imaging mass spectrometry and histology for quantitative biomarker validation on a voxel-by-voxel basis within imaging arrays.